Araştırma Makalesi / Research Article Iğdır Üni. Fen Bilimleri Enst. Der. / Iğdır Univ. J. Inst. Sci. & Tech. 8(1): 255-261, 2018

The Effects of Iron (Fe+3) on The Expression Levels of Heat Stress in Rat Liver (Rattus norvegicus) Tissue

Atena GHOSIGHAREHAGHAJI1, Hamid CEYLAN1, Orhan ERDOĞAN1

ABSTRACT: Iron, one of the most common metals in the world, is important for organisms. It is known that at high concentrations, it damages especially organs such as liver, pancreas, heart. In our study, the effect of iron ion on the expression of the 70 kDa HSP family which small stress was investigated in Rattus norvegicus. In this study, iron ion Fe3 + (0,87ppm, 3ppm, 30ppm, 300ppm) was given to 5 different application groups at different concentrations. At the end of this application period, a cDNA library was formed from the liver tissues taken from the living body. Using these libraries, changes in expression levels occurring in the (Hspa1a, Hspa4, Hspa5) (Hsp90aa1) genes were determined by Real-Time PCR method. Key words: , hsp, iron ion, Rattus norvegicus, real-time PCR. Iğdır Üniversitesi Fen Bilimleri Enstitüsü Dergisi Iğdır Iğdır University Journal of the Institute of Science and Technology Iğdır University Journal of the Institute Science and Technology

Demir İyonunun (Fe3+) Sıçan (Rattus norvegicus) Karaciğer Dokusundaki Isı Stres Proteini (Hsp) Genlerinin Ekspresyon Seviyeleri Üzerine Etkisi

ÖZET: Dünyada en fazla bulunan metallerden birisi olan demir, organizmalar için önemlidir. Yüksek konsantrasyonlarda özellikle karaciğer, pankreas, kalp gibi organlarda hasar oluşturduğu bilinmektedir. Çalışmamızda Rattus norvegicus’da demir iyonunun küçük stress proteinleri olan 70 kDa HSP gen ailesinin ekspresyonu üzerine olan etkisi araştırıldı. Bu çalışma kapsamında 5 farklı uygulama grubuna farklı konsantrasyonlarda demir iyonu Fe3+ (0,87ppm, 3ppm, 30ppm, 300 ppm) verildi. Bu uygulama süreci sonunda canlıdan alınan karaciğer dokularından cDNA kütüpanesi oluşturuldu. Yapılan bu kütüpaneler kullanılarak HSP70 (Hspa1a, Hspa4, Hspa5) HSP90 (Hsp90aa1) genlerinde meydana gelen ekspresyon seviyelerindeki değişimler Real-Time PCR metodu ile tespit edildi. Anahtar Kelimeler: Demir iyonu, gen ekspresyonu, hsp, Rattus norvegicus, real-time PCR

1 Atena GHOSIGHAREHAGHAJI (0000-0002-8856-8287), Hamid CEYLAN (0000-0003-3781-4406),

: 8, Cilt/ Volume Sayı/ Issue : 1, Sayfa/ pp : 255-261, 2018 ISSN: 2146-0574, e-ISSN: 2536-4618 DOI: 10.21597/jist.407883 Orhan ERDOĞAN (0000-0001-8908-7293), Atatürk Üniversitesi, Fen Fakültesi, Moleküler Biyoloji ve Genetik Bölümü, Erzurum, Turkey Sorumlu yazar/Corresponding Author: Orhan ERDOĞAN, [email protected]

Geliş tarihi / Received: 05.09.2017 Kabul tarihi / Accepted: 31.10.2017 Atena GHOSIGHAREHAGHAJI et al.

INTRODUCTION The physiological functions of stress proteins become more important when the cell exposed heat shock. Stress In life, “stress factors” are the internal and external factors that cause physiological balance impairment and proteins prevent cleavage of oligomeric complexes and the “cell stress response” is the response of the cells to opening of polypeptides under heat shrinkage. If re- the stress factors (Jolly and Morimoto , 2000). When folding becomes impossible, it accelerates the removal confronted with stressful situations such as temperature of denatured proteins. On the other hand, the presence of shocks, cells increase the synthesis of a group of proteins denatured proteins in the cell stimulates the production which called heat shock or stress proteins. The stress of stress proteins. Microbial pathogens accelerate the response seen in all living things is shaped by a special gene synthesis of stress proteins to protect the host staphylococci group that is highly conserved in development (Mezquita produced by host phagocytes. If the intracellular pathogen et al., 2001). Apart from its hot application, it is known that Salmonella is previously treated with hydrogen peroxide, stress factors such as heavy metals, free oxygen radicals, the synthesis of stress proteins increases and this protects amino acid analogues stimulate temperature shock it from the effect of hydrogen peroxide in the higher lethal protein synthesis in the cell (Lindquist and Craig, 1988). dose. In general, it has been found that mutants producing In conditions where there is no stress effect, temperature high-dose stress proteins are resistant to heat and oxidant shock proteins are present in the cells, and these proteins, agents at an advanced level, while mutants with defects in which are continuously expressed, are called temperature their stress protein genes are highly sensitive to the killing shock cognates and function in the folding, transporting effect of active macrophages (Zuhal, 2009). and regulating mechanisms of proteins within the cell (Mathew and Morimoto, 1998;Yeğenoğlu, 2007). During temperature stress, DNA synthesis, transcription, RNA MATERIALS AND METHOD processing, translational events, and stops of cell cycle Experimental Animal and Experimental progression. At the same time, denaturation occurs Application in proteins, lysosomal degradation events increase, membrane permeability changes, and there are increases Male Rattus norvegicus strain Sprague-Dawley rats in extracellular ion transport. As a result, transcription were obtained at the Experimental Medical Application and translation of heat-shock protein genes begin. In and Research Center of Atatürk University (Erzurum, such cases the function of heat-shock proteins, such Turkey). The rats used in this experiment were housed as Heat Stress Protein 70 (Hsp70), is to prevent the in a controlled room (22 ± 2 °C) and humidity (40-60%) precipitation of denatured proteins by the function of and in a room which was regularly lighted for 12 hours molecular chaperones, to re-fold these proteins properly in the dark and 12 hours in the desired amount of food and to improve the obtained stress tolerance. Oxidative and water. The rats were left for at least one week in the stress is an important signal for the apoptotic process. period of adaptation with deionized water before starting Changes in the cellular redox state are an effective way the experiment. Animals were exposed to a daily mixture to regulate different apoptotic pathways. HSF-1 transgenic of deionized water and metal for 100 days. During the first mice were found to be more susceptible to oxidative week to the next week, the ion concentration of the water stress (Yan et al., 2002). HSPs act as antioxidants in the was increased incrementally until each group was brought protection of the cellular redox state. Oxygenase is an to its own nutrient concentration. In this experiment, 5 HSP responsible for the production of antioxidants from group of rats were used. The first group used as control bilirubin and biliverdin. It is known that Hsp70 enhances was given only deionized water for 12 weeks. Iron peptide complex stability and peptide binding ability concentration (FeCl3.6H2O) was given in the basic dose under oxidative stress conditions. Redox status in the cell group (1 group; 3 ppm). The first week of deionized affects Hsp70 synthesis. Thus, reduced GSH levels may water, at week 2 and after, 0.87 ppm (in concentrations lead to direct activation of HSF-1 (Marius and Robert, equivalent to the maximum limits determined by WHO) 1996). On the contrary, strong oxidizing agents inhibit iron ion concentration was applied. The third group was the trimerization of HSF-1 by inhibiting its ability to bind treated with deionized water for the first week, 0.87 ppm to DNA. As a result, a moderate change in redox for the second week, and 3 ppm for the third week and homeostasis leads to the activation of HSF-1, while large thereafter. Group 4 was treated with deionized water for 1 changes in redox homeostasis inhibit HSF-1 (Sreedhar week, 0.87 ppm for 2 weeks, 3 ppm for 3 weeks, 30 ppm and Csermely, 2004). concentration for 4 weeks and later. Group 5 was treated

256 Iğdır Üni. Fen Bilimleri Enst. Der. / Iğdır Univ. J. Inst. Sci. & Tech. The Effects of Iron (Fe+3) on The Expression Levels of Heat Stress Protein Genes in Rat Liver (Rattus norvegicus) Tissue with deionized water at week 1, 0.87 ppm at week 2, 3 Semi-quantitative real-time RT-PCR was performed using ppm at week 3, 30 ppm at week 4, 300 ppm concentration the TaqMan Prob. at week 5 and later. No animals died and none of the toxic Primer Design and Synthesis signs that could be seen were seen. All the primers used in this study were synthesized Nucleic Acid Preparation by Shanghai Invitrogen Biotech Co Ltd. (Invitrogen, Liver mRNA expressions of HSP70 (Hspa1a, Hspa4, Shanghai) and these are listed in Table 1. According to the Hspa5), Hsp90 (Hsp90aa1) were evaluated using real- conserved sequences from known testes-specific time RT-PCR analyses. Liver samples were collected from and used to amplify target fragments of the testes-specific 15 rat at the end of day 100 .Total RNA was extracted Hsp70 genes. RNA was used as the gene material and from the liver using trisol, chloroform, isopropyl alcohol, Hspala, Hspa4, Hspa5, Hsp90aa1 were used as primers. 70% ethanol, DPEC (Diethyl pyrocarbonate) according The nucleotide sequences of the mRNA data of NM to the manufacturer’s protocol. The concentration and 031971, NM 153629, NM 013083, and NM 17576102 quality of total RNA were estimated by spectrophotometry were obtained from the gene bank of the internet (absorbance at 260 nm). RNA was reverse transcribed (Anonym, 2012). By the rat Hsp70 and Hsp90 genes to cDNA in a reaction mixture using Transcriptor First (Anonym, 2012). Were used to generate primers specific Strand cDNA Synthesis Kit - Roche. The cDNA was for the maximum of 1000 bp of the genes. The generated then used for Semi-quantitative real-time RT-PCR using primers were checked using specific site specificities Applied Biosystems® 7500 Real-Time PCR Systems. (Anonym, 2012).

Table.1. Primer and prob sequence.

Hspa1a / Forward Primer 5'-ACGCTGGCTGAGAAAGAGG-3'

Hspa1a/ Reverse Primer 5'-’ATCCACCTCCTCGATGGTG -3'

Hspa1a/ TaqMan FAM-TGCACAAGCGGGAGGAGCTG-TAMRA

Hspa4 / Forward Primer 5'-TGCTTGGAAAGGCTGAGTGG-3'

Hspa4/ Reverse Primer 5'-TGGGAATAGAGGGCAATGAG-3'

Hspa4/ TaqMan FAM-TGGGACTGTGGGGCTGGTGC-TAMRA

Hspa5/ Forward Primer 5'-ATTCCTGCGTCGGTGTATTC-3'

Hspa5 /Reverse Primer 5’TGGACGTGAGTTGGTTCTTG-3'

Hspa5/ TaqMan FAM-ACGATCAGGGCAACCGCATC-TAMRA

Hsp90aa1/Forward Primer 5'-CGAGAGCTTGACCGACCCTAG-3'

Hsp90aa1/ Reverse Primer 5'-ATTCCAATGCCAGTATCCAC-3'

Hsp90aa1/ TaqMan FAM-TGGACTCGGGGAAGGAGCTGC-TAMRA

Organ distribution of Hsp70 and Real-time same concentration. Real-time RT-qPCR was performed in RT-qPCR Analysis a C1000™ Thermal Cycler (Applied Biosystems® 7500 The organ-dependent Hsp70 mRNA expression was Real-Time PCR Systems) according to the manufacturer’s measured real-time RT-qPCR as the following method. instructions. The final volume of each RT-qPCR reaction Briefly, first-strand cDNA was prepared as described was 25 μL, which contained 12.5 μL TaqMan prob master above. Gene-specific primers (Q-F and Q-R; Table 1) were mix , 2.5 μL of diluted cDNA template, 8.7 μL of PCR- designed based upon the cloned Hsp70 cDNA to produce grade water, and 0.25 μL of each 10 μM primer. PCR amplicons in different sizes. All RT-qPCR reactions were conditions were as follows: 95 °C for 30 s, followed by 40 performed in triplicate using extracted RNA (pooled) of the cycles of 95 °C for 5 s and a 0.5 °C/5 s incremental increase

Cilt / Volume: 8, Sayı / Issue: 1, 2018 257 Atena GHOSIGHAREHAGHAJI et al. from 60 °C to 95 °C that lasted 30 cycles. Hsp70 expression to be statistically higher at 300 ppm than at the control at levels were calculated by the 2−ΔΔCt comparative Ct method 0.87 ppm, 3 ppm, 30 ppm and 300 ppm treatment groups (Livak and Schmittgen, 2001). Mean and standard (Figure 1.a). There were statistically significant differences deviations were calculated from triplicate experiments, and between the levels of Hspa1a mRNA of liver tissues exposed presented as the n-fold differences in expression relative to to subletal doses of iron heavy metals in rats. In the 0.87 18S rRNA. ppm, 3 ppm, 30 ppm and 300 ppm groups, p˂0.001 = ***, Statistics control and 0.87 ppm were compared and 0.8 ppm and 3 ppm p˂0.01 were found to be significant (Figure 1.b). When All statistics were analyzed by ANOVA method the gene expression of Hspa4 in liver tissue was examined, and variance analysis in SPSS 17.0 package program. it is seen that the differences between 0.87ppm and 30 ppm Duncan multiple comparison test was used to determine control are significant (0.87 ppm and 3 ppm: p˂0.05 = *, the difference between significant group means as a result 30 ppm and 300 ppm: p˃0.05 = ns) (Figure 1.c). When of ANOVA test and variance analysis. A p value of less group comparisons in liver tissue of Rattus norvegicus than 0.05 was considered statistically significant. All values were examined in general, the differences between gene are shown as Mean ± SDM (Mean ± Standard Deviation) expressions of all groups in relation to the control group were (n = 3). found to be significant 0.87 ppm and 3 ppm comparative RESULTS AND DISCUSSION and 0.87ppm and 30ppm comparative p˃0.05=ns, 3ppm and 30ppm compared p˂0.05=* with other groups except Gene Expression Results 3+ these, the change between the gene expression Fe ion Hsp90aa1 gene expression in the liver tissue of rats concentration was found statistically significant compared exposed to different doses of heavy iron metal was found to the control. p˂0.001=*** (Figure 1.d)

- a - - b -

- c - - d -

Figure 1. Gene expression levels of Hsp90, Hspa1a, Hsp4, Hspa5 in the liver are expressed as red stars, which are the p values obtained from Tukey’s Multiple Comparison Test analysis. The number of stars varies from 1 to 3 in proportion according to the degree of importance. *p˂0.05=*, p˂0.01=**, p˂0.001=*** 258 Iğdır Üni. Fen Bilimleri Enst. Der. / Iğdır Univ. J. Inst. Sci. & Tech. The Effects of Iron (Fe+3) on The Expression Levels of Heat Stress Protein Genes in Rat Liver (Rattus norvegicus) Tissue

While many studies have been done on HSP, activity, oxidative stress produces free radicals. these studies are mostly the effects of heavy metals, Oxidative stress increases the expression of Hsp 70 due temperature, saltiness, stock intensity, competition or to damage of liver cells. There are cases where heavy even calorie restriction on the expression of viable HSP metal application reduces Hsp 70 level. For example, genes. The effects of environmentally important heavy in a study of broiler chickens, the supplementation of metals and organochlorines on the transcriptional the organic form of Se, Cr and Zn resulted in low Hsp profiling of genes encoding heat shock cognate 70 70 mRNA expression levels (Yahav 2009).Different (Hsc70) and inducible 70 (Hsp70) temperatures were applied to different fish species, and in the tubercle fibroblast cell line were investigated. in general, an increase in the exoplasmicity of this HSP Specific reverse transcriptase polymerase chain reaction family was recorded (Krone et al., 1997;Palmisano et (RT-PCR) was used to test the effects of heavy metals al., 2000;Mesa et al., 2002;Murtha et al., 2003;Bowen (Cd +2, Cu +2 and Ni +2) and organochlorines (aroclor et al., 2006). Another criterion applied to investigate 1254, hexachlorobenzene and 2-4-dichloroaniline) on the expression of the HSP group is heavy metals and the cell stress response. Hsp70 expression was induced various pollutants, and various concentrations were in fibroblasts when exposed to concentrations of heavy examined by applying to fish groups (Ait-Aissa et al., metals as low as 0.01 μM, whereas expression of Hsc70 2000;Boone and Vijayan 2002;Feng et al., 2003) and expression was induced when exposed to concentrations the increase in HSP expression was recorded in all of as low as 0.001 μM organochlorine. These studies show the applied doses. that gene members of the Hsp70 family are sensitive to In a study with mature zebrafish, the fish were environmental considerations (Deane et al., 2006). The exposed to 37 ° C temperature and RNA was isolated distribution of heavy metals in various tissues of Chanos from different tissues and compared with Hsp expression chanos collected from dirty regions and the associated using RT-PCR. Although Hsp70 expression showed oxidative stress have been studied comparatively to a steady increase in brain, liver, and muscle, Hsp47 the fish collected from the less polluted regions of increased only in the brain, Hsp90 α-β and heat shock Kaattuppalli Island. Concentrations of copper, lead, factor 1 (Hsf1a) were prominent in all three tissues, but zinc, cadmium, manganese and iron were measured in the temperature did not increase in response to stress. gills and liver. It is a highly injured work to prevent In comparison with HSP expression, they observed an the detection of Hsp70 biomarkers for heavy metal increase in basal Hsp70 and Hsf1a levels compared induced oxidative stress and the heavy metal pollution with younger adults. They also noted that there may be that may accumulate in the future (Sivakumar et al., age differences in response to heat shock and mature 2013). In our study, we tried to determine the effect of zebrafish are suitable models for the aging process for HSP gene expression in S. dawley rabbits exposed to Hsp studies (Murtha et al., 2003) Fe + 3 ion. It has been reported that HSP gene expression changes in response to heat shock depending on age In our study, iron ions (0.87 ppm, 3 ppm, 30 and season (Murtha et at., 2003). As a matter of fact, ppm, 300 ppm) were given to Sprague-Dawley male in summer and winter the level of Hsp70 is high in rats of the male Rattus norvegicus strain and the HSP the liver and in the spring (May) this level has fallen gene was monitored for exoplasm and an increase in and the water has been exposed to various pollutants HSP expression of iron ion was observed at different (Köhler et al. 2001). In a study conducted on oysters, concentrations. Densities of the resulting bands were it was reported that Hsp70 was more expressed in the measured and an increase in HSP expression was autumn months (Encomio et al., 2005). The effects of observed due to the increase in iron concentration. heat shock and heavy metals have been extensively According to the obtained results, changes in the studied in all studies with this gene. Due to the various expression levels of the Hsp70 (Hspa1a, Hspa4, metabolic functions of different organs under the Hspa5) and Hsp90 (Hsp90aa1) genes were detected influence of heat stress, the level of Hsp 70 mRNA by Real Time PCR method. HSP90aa1 and Hspa1a expression varies significantly in different tissues. Hsp expression levels of HSP at 30 ppm and 300 ppm in the 70 mRNA expression is higher in tissues such as heart gene expression levels were significantly increased and liver. Because these tissues have high metabolic compared to the control (p<0.001=***). There was

Cilt / Volume: 8, Sayı / Issue: 1, 2018 259 Atena GHOSIGHAREHAGHAJI et al. an increase in Hspa5 and Hspa4 gene expression al., 1992), liver and heart (Locke and Tanguay, 1996). levels when 3 ppm iron concentration was applied. Considering the physical and chemical conditions When group comparisons in liver tissue of Rattus that are effective in HSP expression, it is necessary norvegicus were examined in general, differences to better adjust the ambient conditions to remove the between gene expressions of all groups were found stress factors of the fish from the single environment. to be significant according to the control group. The We studied how iron stress applied on mice affected changes in Hspa5 and Hsp90aa1 gene expression the expression of heat shock proteins. As a result, we levels between 0.87ppm and 3ppm iron ion exposure have examined comparatively which iron concentration groups were compared between 0.87ppm and 30ppm influences which of the HSP genes we work most. iron ion exposure groups (p˃0.05 = ns). A significant correlation was found between the results of 3ppm and 30ppm concentration application (p˂0.05 =*). CONCLUSION The other groups were statistically significant As a result, we observed that among the genes that (p˂0.001 = ***) compared to the control group we examined expression change, the highest increase between the 30 ppm and 300 ppm iron ion-treated and change in the mice exposed to 300 ppm iron iodine Hspa4 gene expression levels compared to the other occurred in the expression of Hspa1a gene. This study groups. Expression of these stress proteins can vary is an important study in determining the effect of stress with many psychological, pathological, and age on the expression of HSP genes in living organisms. factors (Murtha et al., 2003). The work done by these stress potholes, which are molecular protectors in all organisms, is not limited to fish only, but has been ACKNOWLEDGEMENTS tested in all organisms from man to mouse. We would like to thank the Center for Medical Mice exposed 4-7 and 22-28 month old mice Experimental Application and Research (ATADEM) to temperature stress at 42.5 ° C for 30 minutes and affiliated to Atatürk University’s Rectorate, which HSP70 expression was found to be 40-50% less than ensures that animal experiments are carried out in in young mice in the elderly. In another study with accordance with the applicable national guidelines for mice, temperature exposure observed increases in HSP the use and care of laboratory animals and approved by and protein expression with age in neurons (Pardue et the Local Animal Ethics Board of Ataturk University.

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